isolating cranial neural folds from a chick embryo for a neural crest cell culture

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Transfer an embryo to a clean dish containing Ringer&#39;s Pen-Strep solution. Gently swish the embryo back and forth to clear away any yolk that obscures the view, and exchange the Ringer&#39;s Pen-Strep solution or transfer it to a fresh dish if it becomes cloudy. Position the embryo dorsal and frame-side up under a dissecting microscope. Leave the embryo on the paper frame to keep it stretched and held in place.
Then, remove the vitelline membrane using forceps to expose the neural folds. Include tissue caudal to the expanding optic vesicles and rostral to the hindbrain, where rhombomere constrictions are just beginning to appear. Using spring scissors, carefully excise the midbrain neural folds. Take care to excise the dorsalmost aspect of the neural fold with minimal contamination of the neural tube and non-neural ectoderm, and transfer the neural folds to a clean dish containing Ringer&#39;s Pen-Strep solution using a P20 pipettor or a sterile glass Pasteur pipette rinsed with yolky Ringer&#39;s Pen-Strep solution.
Store collected folds on ice while dissecting additional folds. After removing the culture dish from the incubator, use a pipettor or Pasteur pipette to remove the fibronectin solution from coverslips dish or well. After rinsing the fibronectin-coated substrate with Ringer&#39;s Pen-Strep solution, add an appropriate volume of complete culture media to the dish or well.
To block the plastic and prevent the tissue from sticking, use a P20 or P200 pipettor, and rinse the pipette tip with yolky Ringer&#39;s Pen-Strep solution. Then, transfer the isolated neural folds towards the center of the fibronectin-coated coverslip, taking care to transfer as little Ringer&#39;s Pen-Strep solution as possible. After allowing the explants to settle for 10 to 15 minutes, place them into a humidified chamber at 38 degrees Celsius by slowly and carefully carrying the culture dish with plated neural folds.

isolating-cranial-neural-folds-from-chick-embryo-for-neural-crest

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of solutions and materials
<ol>
	<li>Prepare Ringer&#39;s solution by mixing 123.3 mM NaCl, 1.53 mM CaCl2, 4.96 mM KCl, 0.809 mM Na2HPO4, and 0.147 mM KH2PO4 (see Table of Materials). Adjust pH to 7.4 and filter sterilize into 100 mL bottles. Store in a clean, dry location. Do not wash bottles with soap (treat as tissue culture glassware).</li>
	<li>Prepare 1 mg/mL stock solution fibronectin (FN) (see Table of Materials) by dissolving FN in sterile Ringer&#39;s solution and store in 100 &#181;L aliquots at -80 &#176;C (stable for at least 4 years).</li>
	<li>Prepare complete culture media by supplementing L15 media with 0.8% L-glutamine, 0.08% penicillin/streptomycin, 10% FBS, and 10% chick embryo extract. Create 3 mL aliquots and store at -20 &#176;C or -80 &#176;C. 
	NOTE: If cultures are incubated in a CO2 incubator, Dulbecco&#39;s Modified Eagle Medium F12 (DMEM/F12) needs to be substituted for L15, which is buffered for air.</li>
	<li>Prepare filter paper support frames (approximately 1 cm x 1.5 cm rectangles of paper with two or three punched holes overlapping on the long axis).
	<ol>
		<li>Punch holes along the edge of a large piece of filter paper using a standard three-hole punch, cut/trim the punched edge into a strip, and then cut the strip between the holes into rectangles ~ 1.5 cm in length. Autoclave filter papers before use (optional).</li>
	</ol>
	</li>
	<li>Gather dissection tools, including fine forceps (#5), blunt forceps, dissection scissors, spring scissors, and sharpened tungsten needles.</li>
</ol>
2. Preparation of culture dishes
<ol>
	<li>Select neural fold culture dishes (see Table of Materials) appropriate for the downstream application.
	<ol>
		<li>For cultures that will be fixed and stained, use glass coverslips placed into multi-well tissue culture dishes. 
		NOTE: Coverslips that match the well size will move less with fluid mixing, making explants less likely to shift and more likely to adhere to the coverslip rather than the dish bottom.</li>
		<li>Select glass-bottom single chamber or divided chamber dishes (see Table of Materials) for live imaging with an inverted microscope.</li>
		<li>For live imaging on an upright or stereo microscope, use 6-, 12-, or 24-well optically appropriate tissue culture plates.</li>
		<li>Select plastic tissue culture dishes for the mass collection of migratory neural crest cells for high throughput analyses.</li>
	</ol>
	</li>
	<li>Add 10 U/mL of penicillin and 10 &#956;g/mL of streptomycin to a 100 mL bottle of sterile Ringer&#39;s solution (for example, 100 &#956;L of 10,000 U/mL penicillin and 10,000 &#956;g/mL streptomycin, to make Ringer&#39;s P/S). Use Ringer&#39;s P/S within 1 week.</li>
	<li>Thaw an aliquot of 1 mg/mL of FN on ice. Dilute with Ringer&#39;s P/S to a concentration of 10-100 &#956;g/mL of FN.</li>
	<li>Pipet enough FN solution to cover the bottom of the well or dish. For example, 100 &#956;L per well in a 24-well plate, 500 &#956;L for a 35 mm dish.</li>
	<li>Replace the lid and incubate dishes or plates with FN solution in a humidified incubator (or a covered tray with distilled water-soaked paper towels) at 38 &#176;C (100 &#176;F) for at least 1 h while dissecting neural folds.</li>
</ol>
3. Dissecting neural folds
<ol>
	<li>Transfer an embryo to a clean dish containing Ringer&#39;s P/S solution and rinse the embryo by holding the filter paper frame with forceps and gently swishing back and forth to clear away any yolk that obscures the view of the embryo. Exchange the Ringer&#39;s P/S or transfer to a fresh dish if it becomes cloudy.</li>
	<li>Position the embryo dorsal/frame side up under a dissecting microscope. Leaving the embryo on the paper frame to keep it stretched taut and held in place, remove the vitelline membrane using forceps to expose the neural folds. 
	NOTE: If the embryo falls off the paper frame, it can be laid out flat in the dish or pinned to a sylgard coated dish for dissection.</li>
	<li>Using spring scissors or a sharpened tungsten needle, carefully excise the midbrain neural folds. Include tissue caudal to the expanding optic vesicles and rostral to the hindbrain, where rhombomere constrictions are just beginning to appear (the cardiac crescent is also a useful indicator, Figure 1B, C). Take care to excise the dorsal-most aspect of the neural fold with minimal contaminating neural tube and non-neural ectoderm (Figure 1C).</li>
	<li>Transfer the neural folds to a clean dish containing Ringer&#39;s P/S using a P20 pipettor or a sterile, glass Pasteur pipette rinsed with yolky Ringer&#39;s P/S (this blocks the plastic or glass to prevent the tissue from sticking). Store collected folds on ice while dissecting additional folds.</li>
</ol>
4. Plating neural folds
<ol>
	<li>Thaw an aliquot of complete culture media (section 1, step 3). Add 100 U/mL of penicillin and 100 &#181;g/mL of streptomycin and filter sterilize. Keep prepared media at 37-38 &#176;C while performing other steps.</li>
	<li>Remove culture dishes from the incubator (section 2, step 5). Using a pipettor or Pasteur pipette, remove the FN solution from coverslips, dishes, or wells. After rinsing the FN-coated substrate with Ringer&#39;s P/S, add an appropriate volume of complete culture media to the dishes or wells (500 &#181;L for wells in a 24-well plate, 2 mL for a 35 mm dish or a six-well plate). 
	NOTE: Smaller volumes can preserve expensive reagents (e.g., as low as 200 &#181;L for a 24-well plate) but ensure that the dish is sufficiently humidified to avoid evaporation.</li>
	<li>Using a p20 or p200 pipettor, first, rinse the pipette tip with yolky Ringer&#39;s P/S to block the plastic and prevent the tissue from sticking. Then, transfer the isolated neural folds onto the FN coated coverslips, taking care to transfer as little Ringer&#39;s P/S as possible. Place the fold(s) toward the center of the FN-coated coverslip. 
	NOTE: One or a few neural folds can be plated on a 12 mm coverslip in a 19 mm well, up to 50 on a 35 mm plate.</li>
	<li>After allowing the explants to settle for 10-15 min, place them into a humidified chamber at 38 &#176;C (100 &#176;F) by slowly and carefully carrying the culture dishes with plated neural folds. This will minimize the shifting of neural folds in the wells, ensuring they remain dispersed and adhere to any coverslips. 
	NOTE: When using L15 (not DMEM), explant culture dishes can also be incubated in an egg incubator using a covered tray with moistened paper towels.</li>
	<li>Incubate neural fold cultures in the humidified incubator for the duration of active migration (about 16-20 h total, Figure 2).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AxioObserver equipped with an LSM710 confocal scan head controlled by ZEN 3.0 SR software</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used alpha Plan-Apochromat 100x/1.46 Oil DIC M27 objective</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber dishes (glass bottom, single or divided)</td>
			<td>MatTek; Cell Vis</td>
			<td>P35G-1.5-14-C (MatTek) 
			&#160;X000NOJQGX (Cellvis) X000NOK1OJ (Cellvis)</td>
			<td>Single chamber 35 mm or 4 chamber 35 mm</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Carolina Biological Supply Company</td>
			<td>633029, 633031, 633033, 633035, 
			&#160;633037</td>
			<td>circles, 0.13&#8211;0.17 mm thickness, available in 12-25 mm diameter</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>11320033</td>
			<td>Alternative for L15 media</td>
		</tr>
		<tr>
			<td>Egg incubator</td>
			<td>Sportsman</td>
			<td>1502</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Fisher Scientific</td>
			<td>CB-40008A</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td></td>
			<td>grade 3MM chromatography</td>
		</tr>
		<tr>
			<td>Forceps (blunt)</td>
			<td>Fisher Scientific; Thomas Scientific</td>
			<td>08-890 (Fisher);1141W97 (Thomas)</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (fine)</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr>
			<td>Pin holder&#160;</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td>For tungsten needle (alternative for spring scissors)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 media</td>
			<td>Invitrogen</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-148</td>
			<td>10,000 Units/mL Penicillin; 10,000 mg/mL Streptomycin</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>VWR (or similar)</td>
			<td></td>
			<td>60 mm, 100 mm</td>
		</tr>
		<tr>
			<td>Scissors (dissection)</td>
			<td>Fine Science Tools</td>
			<td>14061-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>2.5 mm cutting edge (alternative for tungsten needle)</td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Krayden</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters</td>
			<td>Sigma-Aldrich</td>
			<td>SLGVM33RS</td>
			<td>Millex-GV Syringe Filter Unit, 0.22 &#181;m, PVDF, 33 mm, gamma sterilized</td>
		</tr>
		<tr>
			<td>Tissue culture dishes</td>
			<td>Sarstedt</td>
			<td>83-3900</td>
			<td>35 mm culture dishes for bulk neural fold cultures</td>
		</tr>
		<tr>
			<td>Tungsten wire</td>
			<td>Variety of sources</td>
			<td></td>
			<td>0.01&#34; diameter for tungsten needle (alternative for spring scissors)</td>
		</tr>
	</tbody>
</table>

Source: Jacques-Fricke, B. T., et al. <a href="https://app.jove.com/v/63799">Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.</a> J. Vis. Exp. (2022).
This video illustrates a technique for isolating cranial neural folds from the midbrain region of chick embryos. It presents a detailed dissection process of the neural folds, their placement, and attachment on fibronectin-coated coverslips, followed by the observation of neural crest cell migration from the neural folds.

<img alt="Figure 1" src="/files/ftp_upload/22537/22537_Figure1.jpg" /> 
Figure 1. Dissection of chick dorsal neural folds. Working in Ringer&#39;s P/S, spring scissors were used to excise neural folds. (A) Embryo dorsal view, anterior toward the top of the figure. Neural folds appear more opaque than the surrounding tissue. Midbrain neural folds lie posterior to the optic lobes (pink) and anterior to the cardiac crescent (yellow). (B)</strong...

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Take a chick embryo supported within a filter paper frame and position it under a dissecting microscope.
Remove the vitelline membrane, a protective coating surrounding the embryo.
Identify the midbrain neural folds located posterior to the optic lobes and anterior to the cardiac crescent, the embryonic precursor of the mature heart.
The neural folds contain premigratory neural crest cells, which are multipotent stem cells that transform into migratory cells during development.
These migratory cells differentiate into neuronal and non-neuronal cells.
Excise the dorsal-most aspect of the neural folds and transfer the tissue to a culture dish containing buffer.
Next, place coverslips coated with an adhesion protein in culture plate wells containing media.
Transfer the neural folds onto the coated coverslips. Incubate to facilitate tissue attachment.
During culturing, migratory neural crest cells emerge from the neural folds onto the coated surface, establishing a neural crest cell culture.

22 vues. Isolement des plis neuraux crâniens d’un embryon de poulet pour une culture cellulaire de crête neurale

Vidéo: Isolement des plis neuraux crâniens d’un embryon de poulet pour une culture cellulaire de crête neurale - Expérience

Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

O9-1 is a multipotent mouse neural crest cell line. Here we describe detailed step-by-step protocols for culturing O9-1 cells, differentiating O9-1 cells into specific cell types, and genetically manipulating O9-1 cells by using siRNA-mediated knockdown or CRISPR-Cas9 genome editing.

Culture et la Manipulation des cellules de la crête neurale O9-1

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

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JoVE Journal

Génétique

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Dissection, culture et analyse des cellules neurales primaires de crête neurale de la souris pour l'étude de la délamination et de la migration neurales de cellules de crête

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Biologie du développement

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

In vitro Étude des effets de la matrice extracellulaire riche en hyaluronanes sur la migration des cellules de la crête neurale

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube ...

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

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